The overall goal of this procedure is to identify HIV positive cells using an HIV one rev dependent lentiviral vector carrying the green fluorescent protein. This is accomplished by first producing the rev dependent lentiviral particles by cot transfection. The second step of the procedure is to infect human CD four T cells with HIV one.
Next, the HIV positive human CD four T cells are super infected with the lentiviral particles carrying the GFP gene. The final step is to identify HIV positive cells by measuring GFP positive cells by flow cytometry. Ultimately, results can be obtained that show the percentage of GFP positive cells through flow cytometry.
The implications of this technique extend toward therapy of HIV infection due to the fact that the rev dependent vector can also deliver and selectively express therapeutic genes in HIV infected cells to induce killing of HIV positive cells. Demonstrating the procedure will be gi, a research assistant professor from our laboratory To produce rev dependent lentiviral particles. HEK 2 93 T cells are transfected with three plasmids, the rev dependent GFP lentiviral vector, and HIV one packaging construct and a plasmid carrying the VSVG glycoprotein one day prior to plasmid transfection seed, two times 10 to the six HEK 2 93 T cells in 10 milliliters of medium.
In a 100 millimeter Petri dish. Grow cells overnight at 37 degrees Celsius, 5%carbon dioxide. On the following day, replace the supinate from the cells with five milliliters of fresh medium.
Then continue to culture the cells for four hours at 37 degrees Celsius, 5%carbon dioxide. Prepare transfection reagents before the end of the four hour incubation. The HEK 2 93 T cells will be transfected with the three plasmids at a ratio of 10 micrograms of rev dependent GFP lentiviral vector, 7.5 micrograms of HIV one packaging construct and 2.5 micrograms of VSVG glycoprotein.
First pre-mix the three plasmid DNAs at the desired ratio. Then set up two five milliliter polystyrene tubes. Add 480 microliters of two times HBS buffer to the first tube to the second tube.
Add 60 microliters of two molar calcium chloride buffer the plasma DNAs and the transfection buffer to a total volume of 480 microliters. Next, add the plasma DNA calcium chloride mixture. Dropwise to the two times HPS tube incubated room temperature for 30 minutes.
A fine precipitate will gradually form. Now add all 960 microliters of the mixture dropwise by peppe to the HEK 2 9 3 T cells. Incubate at 37 degrees Celsius, 5%carbon dioxide overnight.
The next day remove the S supernatant and add 10 milliliters of fresh medium. Incubate the cells again at 37 degrees Celsius, 5%carbon dioxide overnight. At 48 hours post transfection, harvest the virus by removing the SUP natant and transferring it into a 50 milliliter sterile tube.
Add 10 milliliters of fresh medium into the Petri dish and continue to culture overnight. Store the virus supinate at four degrees Celsius. Continue to harvest at 72 hours post transfection by collecting the SUP natant and combining it with the previously harvested sup natant.
Then centrifuge the S supernatant at 500 times G for 15 minutes to pellet and remove cell debris. After centrifugation, collect the S supernatant and transfer it into a new tube. Then filter the supinate through a 0.22 micromolar millipore filter.
The virus will be further concentrated through a size exclusion column, which will be demonstrated next to concentrate viral particles. Load the viral supinate into a size exclusion column and centrifuge at 6, 000 times G at four degrees Celsius for 15 minutes. Collect the concentrated viral s supernatant, setting aside about 100 microliters for determining the viral titer.
Aliquot the rest and store it minus 80 degrees Celsius. Determining viral titer is one of the most critical aspects of this procedure. A higher large publicity of infection is needed for a super infection of cells with the GFP VIR vector.
In order to obtain a percentage of GFP positive cells detectable by flow cytometry To determine viral titer. A TNF treated HIV V one positive jerk cat cell line will be infected with the viral supinate. These HIV V one positive cells are cultured.
In a 96 well plate at 2.5 times 10 to the five cells per milliliter in a total volume of 100 microliters. Prepare six serial tenfold dilutions of the rev dependent lentiviral particles for infecting the HIV one positive cells add 100 microliters of each serial dilution of virus to each well of cells. Incubate in a 37 degrees Celsius carbon dioxide incubator for seven days.
The titer of the particle is estimated by observing GFP positive cells under a fluorescent microscope. Calculations are made following the method of Reid and MU and the titer is determined as the dilution point that can result in GFP positive wells. In 50%of wells infected to mark IV one positive cells with the rev dependent lentiviral particles.
A human CD four T cell line will first be infected with HIV one. We use two times 10 to the five cells for each infection. Two hours prior to infection.
Add poly brain to the cells to a final concentration of four micrograms per milliliter. Incubate at 37 degrees Celsius for two hours. Next infect cells by petting the HIV solution into one of the five milliliter tubes.
The other two tubes of cells are not infected and will serve as controls. Incubate all cells at 37 degrees Celsius for two hours. After two hours, centrifuge the cells at 300 times G for 10 minutes, wash away the cell-free virus by discarding the sup, natant and resuspend cells in fresh medium at a concentration of two times 10 to the five cells per milliliter culture at 37 degrees Celsius for 48 hours.
After 48 hours, the HIV infected cells are ready for infection. With the rev dependent lentiviral particles use two times 10 to the five cells for infection at 100 microliters of lentiviral particles to the HIV V one infected cells as a control HIV V one uninfected human CD four T cells are also infected with 100 microliters of lentiviral particles. Incubate the cells at 37 degrees Celsius overnight on the following day.
Replace the spent medium with fresh, medium and resuspend cells at a concentration of two times 10 to the five cells per milliliter. 48 hours post-up infection, pellet the cells by centrifugation and then resuspend in 500 microliters of 1%Para formaldehyde transfer the resuspended cells into a flow cytometry tube for flow cytometry analysis. After a 20 minute incubation at room temperature, the cells can be analyzed on a fax scan analyzer for GFP positivity.
If the experiments are successfully performed, the rev dependent lentiviral vector V-N-L-G-F-P-R-R-E-S-A will permit the highly specific detection of replicating HIV in living cell populations through measurement of green fluorescence protein GFP expression. In this example, harvested C-E-M-S-S cells were stained with a PE labeled rat monoclonal antibody against mouse CD 24 HSA and then analyzed on a flow cytometer for both HSA and GFP expression as shown here. A sizable GFP population was detected in HIV one infected cells super infected with V-N-L-G-F-P-R-R-E-S-A.
In contrast, GFP positive cells were not detected in either HIV V one uninfected C-E-M-S-S cells without lentiviral super infection or HIV one uninfected cells with lentiviral super infection. Don't forget that working with HIV can be extremely hazardous and precautions such as biosafety levels through practices should always be taken while performing this procedure.
